As environmental concerns increase over the production and disposal of conventional petrochemical-based plastics, there is a growing incentive to find a method of producing inexpensive alternatives. Bioplastics have numerous advantages over petrochemical-based plastics. Unlike petrochemical-based plastics, bioplastics rapidly biodegrade and are non-toxic. Bioplastics are derived from renewable resources, decreasing demand for non-renewable petrochemical resources. Bioplastics have lower energy inputs than petrochemical-based plastics, and their production results in lower CO2 emissions than petrochemical plastic production. It is therefore of great interest to find improved methods for producing bioplastics.
Bioplastics include various biopolymers such as polyhydroxyalkanoates (PHA), and particularly the polymer of hydroxybutyrate, polyhydroxybutyrate (PHB). PHAs are polyesters with repeating subunits (100-30,000) that have the formula —[O—CH(R)(CH2)xCO]—.
The most common type of PHA is PHB, where R═CH3 and x=1. Another is polyhydroxy valerate (PHV), where R═CH2CH3 and x=1. PHAs are produced by many bacteria under unbalanced growth conditions when they have access to surplus carbon but lack an essential nutrient, such as phosphorus, nitrogen, sulfur, iron, sodium, potassium, magnesium, or manganese. Under these conditions, the bacteria hoard the carbon, storing it as intracellular PHA granules. The granules are consumed when supplies of carbon and energy become limiting or when the limiting nutrient again becomes available.
The most common known methods of PHA production use pure cultures, relatively expensive fermentable substrates, as sugar from corn, and aseptic operation. The price of PHA produced using this feedstock and methodology currently exceeds the price needed to be competitive with petrochemical-based plastics. Thus, an important challenge is to provide improved methods for producing PHAs that are more efficient and less expensive, so that bioplastics can become commercially competitive with petrochemical-based plastics. Some methanotrophs have been shown to produce PHBs from methane under nutrient limited conditions. The PHB-producing potential of most methanotrophic species, however, remains largely unexplored, as are methods for efficient and inexpensive biosynthesis of PHB.
PHB production is widespread but not universal amongst wild-type methanotrophs. Type II methanotrophs are known to naturally produce PHB while PHB production has not been documented in type I methanotrophs. In mixed culture growth, PHB production is therefore contingent upon selective growth conditions that allow for rapid growth of type II methanotrophs while inhibiting the growth of type I methanotrophs. Currently known selection methods unrelated to nitrogen source manipulation include growth at low pH, growth with no copper in the growth medium, and growth in dilute medium. Continuous growth on ammonium, urea, dinotrogen gas or hydroxylamine has been shown previously. Continuous growth on these nitrogen sources severely limits either growth rates or selectively, while intermittent addition according to the current invention has no such constraints and is therefore a suitable technology for large scale production.
What is needed is a method of selecting Type II methanotrophs from a mixture of Type I and Type II methanotrophic cells, where inhibited growth of the Type I methanotrophic cells and an enhanced growth of the Type II methanotrophic cells forms